The present invention relates to antioxidant compounds, pharmaceutical compositions containing same and their use for preventing or reducing oxidative stress. More particularly, the present invention relates to novel non-central nervous system (CNS) and CNS targeted antioxidants and their use in treating non-CNS and CNS disorders, diseases or conditions associated with a formation of oxidative stress.
Oxidative Stress:
The cellular physiological reduction-oxidation (redox) state, which is dependent on concentrations of oxygen and reactive oxygen species (ROS), is involved in controlling central biochemical regulatory processes, such as tyrosine phosphorylation, regulation of transcription and alteration in messenger RNA stability (1) and it is finely balanced by specific enzymes, such as superoxide dismutase (SOD), catalase, gluthatione peroxidase and thioredoxin, and selective antioxidants, such as glutathione. Regulated homeostasis of the intracellular redox state is essential to the proper physiological functioning of the cell, however, overproduction of (ROS), at levels exceeding the neutralization capacity of cellular antioxidant defenses, generates an oxidative state, termed oxidative stress. Such oxidative stress can lead to oxidative injury via processes such as inflammation, apoptosis and mutagenesis.
Inflammation, a normal physiological process involving limited tissue injury, can be pathogenic if uncontrolled, as under conditions of excessive oxidative stress. In such cases, elevation of ROS, via alterations in expression of redox state-responsive genes, causes the ubiquination and destruction of the NF-κB inhibitory proteins, thereby allowing NF-κB to bind to target gene promoters, a pivotal event in the upregulation of multiple pro-inflammatory cytokines (2). An excess of free radicals has been identified in many diseases associated with inflammation, such as sepsis, multiple sclerosis (MS), stroke, myocarditis and rheumatoid arthritis.
While the development and maintenance of a healthy tissue involves properly regulated apoptosis, interference with this process contributes to various pathologies including tumor promotion, immunodeficiency diseases and neurodegenerative disorders. It has been shown that elevation of the intracellular oxidative state, either via addition of reactive oxygen species (ROS) or depletion of cellular antioxidants, can cause apoptosis (3, 4) and much evidence has accumulated linking oxidative stress to activation of specific enzymes involved in apoptosis.
One such enzyme, essential in the signaling pathway of cytochrome c mediated apoptosis, is c-Jun N-terminal kinase (JNK) which is activated in response to UV radiation, cisplatinum treatment or cellular stress. It has been demonstrated that disruption of JNK protects against UV induced apoptosis, resulting in impairment of the mitochondrial death signaling pathway (5).
In a previous study (6), ROS were shown to play a role as intermediate factors in the pathway of various signal transduction pathways involving thioredoxin, a ubiquitous enzyme in all living cells containing a specific redox-active site. Thioredoxin acts as an inhibitor of oxidative stress induced apoptosis by binding to, and thereby inhibiting, apoptosis signal regulating kinase-1 (ASK1), a protein mediating oxidative stress-induced apoptosis via a redox state responsive domain. However, under conditions of excessive oxidative stress, oxidized thioredoxin dissociates from ASK1, thereby activating it and triggering apoptosis.
Pathologies Associated with Oxidative Stress:
Oxidant injury has been implicated in the pathology of a wide-ranging variety of diseases, including many of major clinical and economic impact, such as cardiovascular, neurological, metabolic, infectious, hepatic, pancreatic, rheumatoid, malignant and immunological diseases, as well as conditions such as sepsis, cataract, amyotrophic lateral sclerosis and congenital diseases such as Down syndrome, multiple organ dysfunction (7) and cystic fibrosis
Described below are some of the most widespread and devastating diseases in which oxidative stress is an etiological factor.
Neurodegenerative Pathologies-Involvement of Inflammation and Oxidative Stress: Evidence has accumulated demonstrating a strong linkage of oxidative stress with pathogenesis of major human neurodegenerative disorders (8-10) including Parkinson's disease (11, 12), Alzheimer's disease (13-15), Creutzfeldt-Jakob disease (16) as well as MS (17).
The different pathological markers characteristic of various neurodegenerative diseases, such as Lewy bodies in Parkinson's disease and amyloid plaques in Alzheimer's disease, indicate different causal factors in the initiation of these diseases. However, there is growing evidence that, once initiated, the progression of a large number of neurodegenerative diseases follows similar cellular pathways. Namely, elevation of the intracellular oxidative state in specific regions of the CNS appears to be an important factor in the etiology of diseases such as Alzheimer's disease, Parkinson's disease, spongiform encephalopathies, degenerative diseases of the basal ganglia, motoneuron diseases and memory loss.
For example, a role for oxidative stress in the pathogenesis of Alzheimer's disease was indicated in a recent analysis of the relationship between β-amyloid protein fragment and oxygen radical formation. This study employed a highly sensitive system, utilizing monitoring blood vessel vasoactive responses, in which β-amyloid-mediated enhancement of phenylephrine-mediated vasoconstriction could be abrogated by pretreatment of blood vessels with SOD, an enzyme which scavenges oxygen free radicals (15). Other studies have shown that oxidative stress and free radical production are linked to the presence of β-amyloid fragment (amino acids 25-35) and likely contribute to neurodegenerative events associated with Alzheimer's disease (18). Further studies have shown extensive RNA oxidation in neurons in Alzheimer's disease and Down's syndrome (13, 14) and genetic evidence for oxidative stress in Alzheimer's disease has also been reported (19, 20).
Evidence of a role for elevated oxidative stress in pathogenesis of MS was provided in studies analyzing the role of metallothioneins, enzymes involved in maintenance of redox homeostasis, in MS or experimental autoimmune encephalomyelitis (EAE) (21, 22), in studies demonstrating increased lipid peroxidation in serum and cerebrospinal fluid of MS patients and in studies demonstrating the role of heme oxygenase-1 (HO-1), a heat shock protein induced by oxidative stress, in the progression of EAE (23).
In the case of scrapies, a type of spongiform encephalopathy occurring in sheep, it was demonstrated that pathogenesis is mediated via microglia cells which respond to prion protein fragment Prp106-126 by increasing oxygen radical production (16).
Diabetes: There is convincing experimental and clinical evidence that the generation of ROS is increased in both types of diabetes and that the onset of diabetes is closely associated with oxidative stress. Recently, it was demonstrated that intracellular content of the oxidant H2O2, visualized with 2′,7′-dichlorofluorescein and quantified by flow cytometry, is increased following treatment with high glucose levels. Concomitant elevation of lactate dehydrogenase activity was detected suggesting that high glucose promotes necrotic cell death through H2O2 formation, which may contribute to the development of diabetic vasculopathy (24). Consistent with these results, a recent study has demonstrated that long-term administration of antioxidants can inhibit the development of the early stages of diabetic retinopathy (25). Other studies carried out with treatment of diabetic rats with antioxidants suggest that diabetes-induced oxidative stress and the generation of superoxide may be partially responsible for the development of diabetic vascular and neural complications (26).
Cataract Formation: A role for oxidant injury in cataract formation was shown in early studies demonstrating that decreased levels of the antioxidant hepatic glutathione-S-transferase (GSH) are associated with increasing opacity of the lens (27). Later studies have shown that in the mammalian lens, intracellular oxidants produced by light induced oxidative processes cause oxidative damage, result in changes in gene expression, and are causally related to cataract formation. It is presently believed that H2O2 is the major oxidant to which the lens is exposed (28).
Infectious Diseases: Harmful levels of oxygen free radicals and nitric oxide (NO) are generated in a diverse range of, and are essential to, the pathogenesis of many types of microbial infections (29). Viral diseases whose pathogenesis is associated with oxidative stress include hepatitis C, AIDS, influenza and diseases caused by various neurotropic agents. In many kinds of viral infections high levels of NO generate highly reactive nitrogen oxide species including reactive oxygen intermediates as well as peroxynitrite, via interaction with oxygen radicals. These species of reactive nitrogen cause oxidant injury as well as mutagenesis via oxidation of various biomolecules. Recent evidence has also demonstrated that oxidative stress induced by NO causes further harm by increasing viral mutation rates and by suppressing type 1 helper T cell function. For example, studies employing the equine influenza virus (EIV) influenza model have shown that viral infection causes cytopathogenic effects and apoptosis as a result of oxidative stress (30). Another study has shown that progression of human hepatitis C virus infection involves triggering of oxidative stress via a mechanism in which the non-structural HCV protein NS5A triggers elevation of ROS in mitochondria, leading to the nuclear translocation and constitutive activation of the pro-inflammatory transcription factors NF-κB and STAT-3 (31).
Neurological Dysfunction Following Cardiac Surgery: Cardiac operations, such as coronary bypass surgery, following multiple infarctions has been shown to significantly increase the risk of neurologic dysfunction, such as impairment of brain function and memory (32-34). Studies have provided evidence that such neurological impairment is associated with oxidative stress (35).
Cardiovascular Diseases: The pathogenesis of major cardiovascular diseases, such as atherosclerosis, hypertension, stroke and restenosis, has been shown to involve oxidative stress. Such oxidant stress in the vasculature causes adverse vessel reactivity, vascular smooth muscle cell proliferation, macrophage adhesion, platelet activation, and lipid peroxidation (36). In the case of atherosclerosis, one of the leading causes of mortality in the developed world, pathogenesis specifically involves inflammation and oxidation of lipoprotein-derived lipids (37).
Recent studies have shown that cerebral ischemia followed by reperfusion leads to elevated oxidative stress (38, 39) and that such oxidative stress can cause damage to genes in brain tissue despite functional DNA repair mechanisms (40). Involvement of such oxidative stress in ischemia-associated pathogenesis was further demonstrated in studies reporting increased infarct size and exacerbated apoptosis in glutathione peroxidase-1 (Gpx-1) knockout mouse brain subjected to ischemia/reperfusion injury (41).
Cancer: Studies have shown that oxidative stress is involved in development of cancers, such as prostate cancer, the most common human malignancy and the second leading cause of cancer deaths among men in Western nations (42).
Thus, the pathogenesis of a very broad variety of diseases involves oxidative stress and, as such, methods of reducing oxidative state may provide an attractive means of treating such diseases.
Prior Art Methods of Treating Disease Via Reduction of Oxidative Stress:
Various prior art methods of treating diseases associated with oxidative stress via reduction of oxidative stress have been attempted and have demonstrated the potential effectiveness of treating disease by restoring redox balance. These have involved either prevention of enzymatic production of ROS by specific inhibitors or introduction of exogenous antioxidants for restoring redox balance.
Diseases of the CNS: To overcome high oxidative stress for the treatment of diseases of the CNS, it is desirable to administer agents capable of reducing oxidative stress into the CNS. However, the CNS is physiologically separated from the rest of the body and from the peripheral blood circulation, by the blood brain barrier (BBB). Since the BBB constitutes a very effective barrier for the passage of agents, such as antioxidants, lacking a selective transporter, such as enzymes or other proteins capable of decreasing oxidative stress, administration of such agents must be via direct injection into the brain or cerebrospinal fluid (CSF). Such a route of administration, however, is unacceptably risky, cumbersome and invasive and thus represents a major drawback for this treatment modality.
One approach has employed administration of the antioxidants vitamin E and vitamin C for treatment of neurological diseases, such as Parkinson's disease (43, 44). Vitamin E was found to be ineffective at decreasing oxidative stress in the substantia nigra and, although capable of crossing the BBB, is trapped in the cell membrane and therefore does not reach the cytoplasm where its antioxidant properties are needed. Vitamin C was shown to cross the BBB to some extent, via a selective transporter, nevertheless it has also been shown to be ineffective in treating neurodegenerative diseases of the CNS.
In another approach, antioxidant compounds characterized by a combination of low molecular weight and membrane miscibility properties for permitting the compounds to cross the BBB of an organism, a readily oxydizable (i.e., reducing) chemical group for exerting antioxidation properties and a chemical make-up for permitting the compounds or their intracellular derivative to accumulate within the cytoplasm of cells, have been employed to treat pathology, including CNS pathology, associated with oxidative stress (44).
Diseases of Non-CNS Tissues:
Systemic Administration of Antioxidants: The major prior art approach used for reducing oxidative stress in non-CNS tissues has employed administration antioxidants.
The antioxidant NAC has been employed to treat canine kidney cells so as to attenuate EIV-induced cytopathic effect and apoptosis (30) and to treat atherosclerosis and restenosis following angioplasty (46). Dimers of NAC have also been employed for treating atherosclerosis (37).
The sulphur-containing fatty acid with antioxidant properties, tetradecylthioacetic acid, has been employed to achieve long-term reduction of restenosis following balloon angioplasty in porcine coronary arteries (47).
The antioxidants pyrrolidine dithiocarbamate (PDTC) and NAC have been used to prevent pathogenic HCV mediated constitutive activation of the pro-inflammatory transcription factor STAT-3 (31).
Synthetic antioxidants have also been employed to treat oxidative stress related disease. For example, treatment of asthma has been attempted by reducing the levels of free oxygen using the synthetic reactive oxygen inhibitor 2,4-diaminopyrrolo-2,3-dipyrimidine (48).
Apoptosis in an ischemic swine heart model has been treated with ebselen, a glutathione peroxidase mimic (35).
The cytosolic antioxidant, copper/zinc superoxide dismutase, has been employed to treat blood-brain barrier disruption and infarction following cerebral ischemia-reperfusion (49). Attenuation of ischemia-induced mouse brain injury has been attempted by administration of SAG, a redox-inducible antioxidant protein (50).
Administration of Metabolic Regulators of Antioxidants: Another approach has attempted to employ metabolic regulators of antioxidants to reduce oxidative stress.
One study has attempted prevention of cataract in a chick embryo model via administration of thyroxine to drive metabolic maintenance of hepatic GSH levels so as to reduce oxidative stress induced by glucocorticoids (51)
Hemin, an inducer of the oxidative stress induced protein, heme oxygenase-1, has been utilized to inhibit progression of EAE (23).
Administration of corticosteroids has been employed to treat lipid peroxidation in MS patients (24).
Stimulation of production of the endogenous antioxidant reduced glutathione has been attempted for treating acute respiratory distress syndrome (ARDS), a condition characterized by overproduction of oxidants or ROS by the immune system, by administration of the drug pro-cysteine (Free Radical Sciences Inc., CA, U.S.). This drug functions by boosting cellular production of glutathione by upregulation of cellular cysteine uptake.
A common feature characterizing all of the above described and other antioxidant compounds is their limited diversity in structure, body distribution, cellular distribution, organelle distribution, and/or antioxidant properties, etc. As such, any given antioxidant may prove useful for some applications, yet less or non-useful for other applications. In some cases, a specific antioxidant may efficiently reduce oxidative stress in some body parts, some cells, or some subcellular structures, yet not in others.
There is thus, a great need for, and it would be highly advantageous to have, an antioxidant compound which is devoid of the above limitations, which compound will by hydrolyzed in vivo to a plurality of different antioxidant species which will act in concert to reduce or prevent oxidative stress in a plurality of tissues, cell types and cellular organelles, so as to combat disease, syndromes and conditions associated with formation of oxidative stress, both in non-CNS and CNS tissues.